Metamorph version 4

Immunofluorescence staining of frozen sections on slides was performed by the previously described method (Prasad et al., 2016 (link)). After staining, images were captured with a fluorescent microscope (Axiophot II; Zeiss, Jena, Germany) equipped with a cooled AxioCam HRc CCD camera (Zeiss) and MetaMorph version 4.6.5 software (Molecular Devices).

Frozen sections in slides were de-paraffinized and rehydrated through a graded alcohol series. Antigen retrieval was performed by boiling the slides for 20 minutes in antigen retrieval solution (10 mM sodium citrate, 0.05% Tween-20, pH 6.0). Slides were cooled for 30 minutes at room temperature, rinsed briefly in PBS, circled with a PAP pen and blocked with 1% normal goat serum. The sections were incubated with primary antibodies followed by their respective secondary antibodies (anti-rabbit IgG Alexa Fluor 610 and anti-mouse IgG Alexa Fluor 488). Slides were mounted using Vectashield Mounting Medium with DAPI (Vector Labs) viewed with a Nikon FXA fluorescence microscope equipped with Photometrics Cool Snap CF color camera (Nikon, Lewisville, TX) and MetaMorph version 4.6.5 software (Molecular Devices). The stored images were processed using NIH ImageJ software and Adobe Photoshop.

For immunohistochemical staining, tissue sections were incubated with antibodies against superoxide dismutase 2 (Abcam) for 2 hours. The staining was detected using the streptavidin–biotin peroxidase complex method with the DAB Peroxidase Substrate Kit (SK-4100; Vector Laboratories, Burlingame, CA, USA), and counterstained with hematoxylin. For fluorescence staining, frozen-section tissue slides were fixed and blocked, and then slides were triple stained with: mouse antibody against human beta-2-microglobulin (hβ2M; Abcam) followed by DyLight 488-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Sacramento, CA, USA); rabbit antibody against human IgG (Abcam), human/rat sex determining region Y-box 9 (Sox-9; Abcam), or human/rat P450scc (Abcam) followed by DyLight 594-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) at room temperature for 30 minutes; and 4,6-diamidino-2-phenylindole (Santa Cruz) for the nucleus. All samples were assessed under a fluorescence microscope (Leica Microsystem, Wetzlar, Germany). Images were acquired using MetaMorph version 4.6 (Molecular Devices, Sunnyvale, CA, USA).